This invention relates to a method enabling the immunoassay of a single protein species in a family of proteins or of proteinic complexes having a common antigen determinant but of different sizes.
The method according to the invention enables the successive performance, in a single operation using a novel electrophoretic system of a separation of the proteins according to their size in a controlled cross-linked medium, and an assay by electroimmunodiffusion according to the method of Laurell, C. B. Anal. Biochem. 15 45-52 (1966). The controlled cross-linked medium in which prior electrophoretic migration is carried out enables the blocking of the proteins of size greater than that of the protein to be assayed, so that these proteins cannot interfere in the subsequent assay by electroimmunodiffusion.
It is a particular object of the invention to provide a method of assay of apolipoprotein B (denoted below apo-B) occurring in low density lipoproteins (denoted below LDL) in the serum.
The apolipoprotein-B (apo B) is a constituent protein of different serum lipoprotein complexes: the very low density lipoproteins (VLDL), the intermediate density lipoproteins (IDL) and the low density lipoproteins (LDL). The sizes, chemical compositions, structures and physiopathological roles of these complexes are very different.
Goldstein J. L. and Brown M. S., Ann. Rev. Biochem. 46 897-930 (1977) have shown that apo B of LDL regulates the cell metabolism of the seric lipoproteins and have suggested that it would play a key role in the occurence of the atherosclerotic process. Recent clinical studies (Sniderman A, et al., Proc. Nat. Acad. Sci. U.S.A. 77 604-608 (1980) have confirmed these experimental data by showing the discriminating character of the serum concentration of apo B of the LDLs in the diagnosis and the prognosis of coronopathies.
It is to be noted in addition that the LDLs form a heterogeneous molecular family in which it is possible to individualize several fractions (Krauss R. M. and Burke D. J. J. Lipid. Res. 23 97-104 (1982)) and particularly the lipoprotein lpa of particular constitution and of which the responsibility in the early development of atherosclerosis has been suspected (Kostner G. M. et al. Atherosclerosis 38 51-61 (1981)).
The assay methods of apo B at present proposed all determine the total serum apo B without differentiating the apo B according to its origin and hence its physiological significance. Determination of apo B of different lipoprotein fractions has indeed been proposed by Sniderman et al. (cited above), but it involves the separation of the lipoproteins by preparative ultracentrifugation, a long, expensive and not really quantitative method.